Our laboratory is elucidating the nature of the immune response to murine tumors and establishing principles which may enhance our ability to immunize patients against known tumor antigens. We have used therapeutic cytotoxic T-lymphocyte lines to identify tyrosinase-related protein 2 (TRP-2) as a tumor T-cell-antigen from the B16 melanoma by immuno-screening a cDNA library from this tumor. In a parallel system, we also found that the transmembrane component of a retroviral envelope protein, encoded by a C57BL/6 germline gene was a tumor-associated T-cell antigen for a large number of murine tumors of different histologies (colon adenocarcinoma, fibrosarcoma and melanoma). For each of these tumor antigen models, we have also defined the minimal determinant peptide recognized and used it to generate CTL which were therapeutic for the parental tumor in vivo in adoptive cellular transfer experiments. Subsequent studies defined the nature of the CTL responses to these minimal determinants in naive and tumor-immune animals and described CTL avidity for the peptide-MHC complex as the prime discriminant of anti-tumor immunity in these systems. Experiments showed that in vitro stimulating peptide concentrations modulated the avidity of the CTL generated and that range of CTL avidities in tumor-immunized mice was higher than in naive and other non-immune mice. Continuing work on murine pre-clinical tumor antigen models has also identified a novel protein expressed by the MC38 murine colon carcinoma, named CCA (colon cancer antigen) as an antigen recognized by a T-cell clone with specific tumor reactivity. We have determined the Db-restricted minimal determinant in CCA which is recognized. Successful tumor immunization with whole tumor generates high avidity T-cell responses to CCA in mice, but currently, recombinant viral and peptide vaccinations vs. the whole gene product or the minimal determinant peptide do not prevent tumor growth. Future studies will be directed toward generating high-avidity CTL and tumor protection with CCA-based vaccines and combining multiple antigen targets for one tumor using several of our newly defined antigens.In other studies, we developed methodologies for identifying tumor-specific CTL from patients with renal cell cancer and successfully used these techniques to identify an RCC-associated T-cell antigen. Unmutated FGF-5 was recognized in an HLA-A3 restricted fashion by RCC TIL from a patient showing spontaneous tumor regression). FGF-5 was expressed in 60% of RCC lines and in some other breast and prostate cancer lines which also were immunolgically recognized when transduced with HLA-A3. We are trying to identify specific epitopes for FGF-5 for vaccine development. We have also generated new tumor-specific T-cell lines from this patient (which do not recognize FGF-5) as well as other patients who have shown significant immune-mediated tumor regressions. We are currently cloning these antigens using expression cloning techniques and CTL screening methods developed and refined in the Surgery Branch.In addition to the above work, clinical studies investigating the optimal IL-2 regimen for inducing the regression of advanced renal cell cancer continue. A prospective randomized 3-arm trial comparing subcutaneous IL-2 versus high and low- dose intravenous IL-2 in the treatment of metastatic renal cell cancer is nearing its full accrual of 400 patients. Future trial will concentrate on vaccination against FGF-5 determinants for patients with FGF-5-expressing tumors as well as phase I trials of vaccination using dendritic cells and renal cancer cells or lysates.